Open Access

Complete genome sequence of Thermobispora bispora type strain (R51T)

  • Konstantinos Liolios
  • , Johannes Sikorski
  • , Marlen Jando
  • , Alla Lapidus
  • , Alex Copeland
  • , Tijana Glavina
  • ,  Del Rio
  • , Matt Nolan
  • , Susan Lucas
  • , Hope Tice
  • , Jan-Fang Cheng
  • , Cliff Han,
  • , Tanja Woyke
  • , Lynne Goodwin,
  • , Sam Pitluck
  • , Natalia Ivanova
  • , Konstantinos Mavromatis
  • , Natalia Mikhailova
  • , Olga Chertkov
  • , Cheryl Kuske
  • , Amy Chen
  • , Krishna Palaniappan
  • , Miriam Land,
  • , Loren Hauser,
  • , Yun-Juan Chang,
  • , Cynthia D. Jeffries,
  • , John C. Detter,
  • , Thomas Brettin,
  • , Manfred Rohde
  • , Markus Göker
  • , James Bristow
  • , Jonathan A. Eisen,
  • , Victor Markowitz
  • , Philip Hugenholtz
  • , Hans-Peter Klenk
  • and Nikos C. Kyrpides
Corresponding author

DOI: 10.4056/sigs.962171

Received: 15 June 2010

Published: 30 June 2010

Abstract

Thermobispora bispora (Henssen 1957) Wang et al. 1996 is the type species of the genus Thermobispora. This genus is of great interest because it is strictly thermophilic and because it has been shown for several of its members that the genome contains substantially distinct (6.4% sequence difference) and transcriptionally active 16S rRNA genes. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the second completed genome sequence of a member from the suborder Streptosporangineae and the first genome sequence of a member of the genus Thermobispora. The 4,189,976 bp long genome with its 3,596 protein-coding and 63 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.

Keywords:

Two distinct 16S rRNA genesstrictly thermophilicnon-pathogenicStreptosporangineaeGEBA

Introduction

Strain R51T (= DSM 43833 = ATCC 19993 = JCM 10125) is the type strain of the species Thermobispora bispora, which is the type species of the genus Thermobispora [1]. The generic name of the genus derives from the Greek words ‘thermos’, ‘bis’, and ‘spora’, to indicate high temperature two-spored organisms [1]. Strain R51T was isolated from decaying manure in Berlin (Germany) in 1954 [2]. Other strains were isolated during the same research project from other types of manure in other cities in Germany and in Finland [2]. As deduced from 16S gene sequences, T. bispora was also found in compost in Sweden [3]. Historically, strain R51T was originally classified in 1957 as Thermopolyspora bispora [2]. At the same time, a morphologically similar genus, Microbispora, was described [4], which has priority and T. bispora was subsequently transferred to the genus Microbispora [5,6]. However, based on thermal preferences [2,7], chemotaxonomic features [7], and the two-dimensional polyacrylamide gel electrophoresis patterns of the ribosomal protein AT-L30 [8], Microbispora bispora was subsequently removed from the genus Microbispora to be the type species of the new genus Thermobispora [1]. T. bispora is currently the only species in the genus Thermobispora [1]. In 1997 T. bispora gained interest, as it was described as the first organism to have two distinct (6.4% of total nucleotides) types of transcriptionally active 16S rRNA genes (GenBank accessions U83909 and U83912) [9]. Based on the two copies of the 16S rRNA genes that match best to sequence U83909 the closest related type strain (9% sequence difference [10]) is Micromonospora pattaloongensis [11] of the family Micromonosporaceae; based on the two copies of the 16S rRNA genes that match best to sequence U83912 the closest related type strain (8% sequence difference [10]) is Planotetraspora silvatica [12] of the family Streptosporangiaceae. Neither fit to the taxonomic position as shown in the List of Procaryotic names with Standing in Nomenclature that shows the genus Thermobispora as a member of the family Pseudonocardiaceae, reflecting the current uncertainty of the taxonomic position of T. bispora [13]. In their recent review of Actinobacteria taxonomy, Zhi et al. [14] suggested to place Thermobispora in the suborder Streptosporangineae without assignment to a family, which is in accordance with our SSU rRNA tree (Figure 1). 16S rRNA sequences from environmental samples and metagenomic surveys with both 16S rRNA sequences detected phylotypes with approximately 89-92% 16S rRNA gene sequence similarity to both (U83909 and U83912) reference sequences only in a compost metagenome [21], indicating a very rare occurrence of Thermobispora-spp. in the environment (status March 2010). Here we present a summary classification and a set of features for T. bispora R51T, together with the description of the complete genomic sequencing and annotation.

Figure 1

Phylogenetic tree highlighting the position of T. bispora R51T relative to the type strains of the other genera within the suborder Streptosporangineae (except for Actinoallomurus, which was published after the analysis was completed). The tree was inferred from 1,371 aligned characters [15,16] of the 16S rRNA gene sequence under the maximum likelihood criterion [17] and rooted in accordance with the current taxonomy [18]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [19] are shown in blue, published genomes in bold, e.g. the recently published GEBA genome from Streptosporangium roseum [20].

Classification and features

Figure 1 shows the phylogenetic neighborhood of for T. bispora R51T in a 16S rRNA based tree. The sequences of the four 16S rRNA gene copies in the genome differ from each other by up to 94 nucleotides, and differ by up to 95 nucleotides from the previously published 16S rRNA sequence generated from ATCC 19993 (U58523).

T. bispora cells form substrate mycelia whose hyphae are 0.5 to 0.8 µm in diameter [1] (Figure 2). The aerial mycelia branch monopodally and bear longitudinal pairs of spores [1] (not visible in Figure 2). The spore diameters are usually 1.2 to 2.0 µm, but in liquid media spores with a diameter of 3 µm may occur [1]. The aerial mycelia are white, and the substrate mycelia are yellow or yellowish brown on the media used in the respective study (International Streptomyces Project medium 4 agar and IF0328 agar; Institute for Fermentation) [1]. No soluble pigment is produced [1]. T. bispora is an obligately thermophilic organism (Table 1) [1]. Starch is not hydrolyzed; inositol and rhamnose are utilized for growth, but arabinose and glycerol are not utilized [1]. Also, T. bispora is negative for iodinin production and nitrate reduction [1].

Figure 2

Scanning electron micrograph of T. bispora R51T

Table 1

Classification and general features of T. bispora R51T according to the MIGS recommendations [22]

MIGS ID

       Property

     Term

    Evidence code

       Current classification

     Domain Bacteria

    TAS [23]

     Phylum ‘Actinobacteria

    TAS [13]

     Class Actinobacteria

    TAS [24]

     Subclass Actinobacteridae

    TAS [14,24]

     Order Actinomycetales

    TAS [14]

     Suborder Streptosporangineae

    TAS [14]

     Family Incertae sedis

    TAS [14]

     Genus Thermobispora

    TAS [1]

     Species Thermobispora bispora

    TAS [2]

     Type strain R51

    TAS [5]

       Gram stain

     positive

    TAS [1]

       Cell shape

     mycelia with hyphae

    TAS [2]

       Motility

     non-motile

    TAS [1]

       Sporulation

     sporulating

    TAS [1]

       Temperature range

     thermophile, 50°C - 65°C

    TAS [1]

       Optimum temperature

     not determined

    TAS [1]

       Salinity

     not determined

    TAS [1]

MIGS-22

       Oxygen requirement

     aerobic

    TAS [1,2]

       Carbon source

     inositol and rhamnose

    TAS [1]

       Energy source

     sugars

    TAS [1]

MIGS-6

       Habitat

     compost and other decaying material

    TAS [2,3]

MIGS-15

       Biotic relationship

     unknown

MIGS-14

       Pathogenicity

     not reported

       Biosafety level

     1

    TAS [25]

       Isolation

     decaying mixed manure

    TAS [2]

MIGS-4

       Geographic location

     Berlin, Germany

    TAS [2]

MIGS-5

       Sample collection time

     September 30, 1954

    TAS [2]

MIGS-4.1MIGS-4.2

       Latitude       Longitude

     52.52     14.42

    NAS

MIGS-4.3

       Depth

     not reported

MIGS-4.4

       Altitude

     not reported

Evidence codes - IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [26]. If the evidence code is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in the acknowledgements.

Chemotaxonomy

The cell wall of strain R51T contains predominantly the menaquinone MK-9(H0) (75%) and only small amounts of MK-9(H2) and MK-9(H4) [7]. Strain R51T has a type PIV phospholipid pattern, and contains phosphatidylethanolamine but not phosphatidylglycerol and trace amounts of glucosamine-containing phospholipids [7]. The cell wall contains a major amount of meso-diaminopimelic acid, and the whole-cell hydrolysate contains madurose and galactose [1]. The fatty acid composition of strain R51T is dominated by saturated acids, with iso-C16:0 (55%) being the most frequent acid, followed by anteiso-C17:0 (8%), the unsaturated C18:1 (8%), iso-C18:0 (6%) and C16:0 [7]. Also, strain R51T contains minor amounts of 10-methyl-branched chain fatty acids [7].

Genome sequencing and annotation

Genome project history

This organism was selected for sequencing on the basis of its phylogenetic position [27], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [28]. The genome project is deposited in the Genome OnLine Database [19] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Table 2

Genome sequencing project information

MIGS ID

     Property

     Term

MIGS-31

     Finishing quality

     Finished

MIGS-28

     Libraries used

     Three genomic libraries:     two Sanger libraries – 8 kb pMCL200 and fosmid pcc1FOS     and one 454 pyrosequece standard library

MIGS-29

     Sequencing platforms

     ABI3730, 454 GS FLX

MIGS-31.2

     Sequencing coverage

     7.1× Sanger; 1.1× pyrosequence pseudo-reads

MIGS-30

     Assemblers

     Newbler version 1.1.02.15, phrap

MIGS-32

     Gene calling method

     Prodigal, GenePRIMP

     INSDC ID

     CP001874

     Genbank Date of Release

     May 17, 2010

     GOLD ID

     Gc01281

     NCBI project ID

     469371

     Database: IMG-GEBA

     2501651196

MIGS-13

     Source material identifier

     DSM 43833

     Project relevance

     Tree of Life, GEBA

Growth conditions and DNA isolation

T. bispora strain R51T, DSM 43833, was grown in DSMZ medium 84 (Rolled oats mineral medium) [29] at 55°C. DNA was isolated from 1-1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with lysis modification st/FT according to Wu et al. [28].

Genome sequencing and assembly

The genome of T. bispora was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at Web Site. 454 pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,798 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the parallel phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [30]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. A total of 1,181 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. The final assembly consists of 40,290 Sanger and 1.1× pyrosequence based pseudo-reads. Together Sanger reads and pseudo-reads provided 8.19× coverage of the genome.

Genome annotation

Genes were identified using Prodigal [31] as part of the Oak Ridge National Laboratory genome an-notation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [32]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes Expert Review (IMG-ER) platform [33].

Genome properties

The genome is 4,189,976 bp long and comprises one main circular chromosome with an overall GC content of 72.4% (Table 3 and Figure 3). Of the 3,659 genes predicted, 3,596 were protein-coding genes, and 63 RNAs; fifty pseudogenes were also identified. The majority of the protein-coding genes (71.9%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Table 3

Genome Statistics

Attribute

Value

  % of Total

Genome size (bp)

4,189,976

  100.00%

DNA coding region (bp)

3,548,135

  84.68%

DNA G+C content (bp)

3,034,765

  72.43%

Number of replicons

1

Extrachromosomal elements

0

Total genes

3,659

  100.00%

RNA genes

63

  1.72%

rRNA operons

3

Protein-coding genes

3,596

  98.28%

Pseudo genes

50

  1.37%

Genes with function prediction

2,632

  71.93%

Genes in paralog clusters

491

  13.42%

Genes assigned to COGs

2,610

  71.33%

Genes assigned Pfam domains

2,844

  77.73%

Genes with signal peptides

795

  21.73%

Genes with transmembrane helices

864

  23.61%

CRISPR repeats

6

Figure 3

Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.

Table 4

Number of genes associated with the general COG functional categories

Code

   value

     %age

    Description

J

   149

     5.0

    Translation, ribosomal structure and biogenesis

A

   1

     0.0

    RNA processing and modification

K

   304

     10.3

    Transcription

L

   141

     4.8

    Replication, recombination and repair

B

   1

     0.0

    Chromatin structure and dynamics

D

   21

     1.0

    Cell cycle control, cell division, chromosome partitioning

Y

   0

     0.0

    Nuclear structure

V

   48

     1.6

    Defense mechanisms

T

   192

     6.5

    Signal transduction mechanisms

M

   140

     4.7

    Cell wall/membrane biogenesis

N

   3

     0.1

    Cell motility

Z

   0

     0.0

    Cytoskeleton

W

   0

     0.0

    Extracellular structures

U

   29

     1.0

    Intracellular trafficking, secretion, and vesicular transport

O

   98

     3.3

    Posttranslational modification, protein turnover, chaperones

C

   204

     6.9

    Energy production and conversion

G

   221

     7.5

    Carbohydrate transport and metabolism

E

   279

     9.4

    Amino acid transport and metabolism

F

   82

     2.8

    Nucleotide transport and metabolism

H

   146

     4.9

    Coenzyme transport and metabolism

I

   133

     4.5

    Lipid transport and metabolism

P

   138

     4.7

    Inorganic ion transport and metabolism

Q

   85

     2.9

    Secondary metabolites biosynthesis, transport and catabolism

R

   351

     11.9

    General function prediction only

S

   191

     6.5

    Function unknown

-

   1,049

     28.7

    Not in COGs

Declarations

Acknowledgements

We would like to gratefully acknowledge the help of Susanne Schneider (DSMZ) for DNA extraction and quality analysis. This work was performed under the auspices of the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, and Oak Ridge National Laboratory under contract DE-AC05-00OR22725, as well as German Research Foundation (DFG) INST 599/1-1 and SI 1352/1-2.


This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

References

  1. Wang Y, Zhang Z and Ruan J. A proposal to transfer Microbispora bispora (Lechevalier 1965) to a new genus, Thermobispora gen. nov., as Thermobispora bispora comb. nov. Int J Syst Bacteriol. 1996; 46:933-938 View ArticlePubMed
  2. Henssen A. Beiträge zur Morphologie und Systematik der thermophilen Actinomyceten. Arch Microbiol. 1957; 26:373-414 View Article
  3. Steger K, Jarvis A, Vasara T, Romantschuk M and Sundh I. Effects of differing temperature management on development of Actinobacteria populations during composting. Res Microbiol. 2007; 158:617-624 View ArticlePubMed
  4. Nonomura H, Ohara I. On a new actinomycete, Microbispora, isolated from soil. Abstract 98, pages 31 and 32, Abstracts of papers presented at the annual meeting of the Agricultural Chemical Society of Japan. Tokyo University, April 9th to llth, 1957. Published by the Agricultural Chemical Society (in Japanese). 1957.
  5. Henssen A and Schnepf E. Zur Kenntnis thermophiler Actinomyceten. Arch Microbiol. 1967; 57:214-231 View Article
  6. Lechevalier HA. Priority of the generic name Microbispora over Waksmania and Thermopolyspora. Int Bull Bacteriol Nomencl Taxon. 1965; 15:139-142 View Article
  7. Miyadoh S, Amano S, Tohyama H and Shomura T. A taxonomic review of the genus Microbispora and a proposal to transfer two species to the genus Actinomadura and to combine ten species into Microbispora rosea. J Gen Microbiol. 1990; 136:1905-1913PubMed
  8. Ochi K, Haraguchi K and Miyadoh S. A taxonomic review of the genus Microbispora by analysis of ribosomal protein AT-L30. Int J Syst Bacteriol. 1993; 43:58-62 View ArticlePubMed
  9. Wang Y, Zhang Z and Ramanan N. The actinomycete Thermobispora bispora contains two distinct types of transcriptionally active 16S rRNA genes. J Bacteriol. 1997; 179:3270-3276PubMed
  10. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK and Lim YW. EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences. Int J Syst Evol Microbiol. 2007; 57:2259-2261 View ArticlePubMed
  11. Thawai C, Tanasupawat S and Kudo T. Micromonospora pattaloongensis sp. nov., isolated from Thai mangrove forest. Int J Syst Evol Microbiol. 2008; 58:1516-1521 View ArticlePubMed
  12. Tamura T and Sakane T. Planotetraspora silvatica sp. nov.,and emended description of the genus Planotetraspora. Int J Syst Evol Microbiol. 2004; 54:2053-2056 View ArticlePubMed
  13. Garrity GM, Holt JG. The Road Map to the Manual. In: Garrity GM, Boone DR, Castenholz RW (eds), Bergey's Manual of Systematic Bacteriology, Second Edition, Volume 1, Springer, New York, 2001, p. 119-169.
  14. Zhi XY, Li WJ and Stackebrandt E. An update of the structure and 16S rRNA gene sequence-based definition of higher ranks of the class Actinobacteria, with the proposal of two new suborders and four new families and emended descriptions of the existing higher taxa. Int J Syst Evol Microbiol. 2009; 59:589-608 View ArticlePubMed
  15. Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol. 2000; 17:540-552PubMed
  16. Lee C, Grasso C and Sharlow MF. Multiple sequence alignment using partial order graphs. Bioinformatics. 2002; 18:452-464 View ArticlePubMed
  17. Stamatakis A, Hoover P and Rougemont J. A rapid bootstrap algorithm for the RAxML web servers. Syst Biol. 2008; 57:758-771 View ArticlePubMed
  18. Yarza P, Richter M, Peplies J, Euzeby JP, Amann R, Schleifer KH, Ludwig W, Glöckner FO and Rossello-Mora R. The All-Species Living Tree project: A 16S rRNA-based phylogenetic tree of all sequenced type strains. Syst Appl Microbiol. 2008; 31:241-250 View ArticlePubMed
  19. Liolios K, Chen IM, Mavromatis K, Tavernarakis N, Hugenholtz P, Markowitz VM and Kyrpides NC. The Genomes On Line Database (GOLD) in 2009: status of genomic and metagenomic projects and their associated metadata. Nucleic Acids Res. 2010; 38:D346-D354 View ArticlePubMed
  20. Nolan M, Sikorski J, Jando M, Lucas S, Lapidus A, Glavina Del Rio T, Chen F, Tice H, Pitlick S and Cheng JF. Complete genome sequence of Streptosporangium roseum type strain (NI 9100T). Stand Genomic Sci. 2010; 2:29-37 View Article
  21. Allgaier M, Reddy A, Park JI, Ivanova N, D'haeseleer P, Lowry S, Sapra R, Hazen TC, Simmons BA and VanderGheynst JS. et al. Targeted discovery of glycoside hydrolases from a switchgrass-adapted compost community. PLoS ONE. 2010; 5:e8812 View ArticlePubMed
  22. Field D, Garrity G, Gray T, Morrison N, Selengut J, Sterk P, Tatusova T, Thomson N, Allen MJ and Angiuoli SV. The minimum information about a genome sequence (MIGS) specification. Nat Biotechnol. 2008; 26:541-547 View ArticlePubMed
  23. Woese CR, Kandler O and Wheelis ML. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA. 1990; 87:4576-4579 View ArticlePubMed
  24. Stackebrandt E, Rainey FA and Ward-Rainey NL. Proposal for a new hierarchic classification system, Actinobacteria classis nov. Int J Syst Bacteriol. 1997; 47:479-491 View Article
  25. Classification of bacteria and archaea in risk groups. TRBA 466.Web Site
  26. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS and Eppig JT. Gene Ontology: tool for the unification of biology. Nat Genet. 2000; 25:25-29 View ArticlePubMed
  27. Klenk HP and Göker M. En route to a genome-based classification of Archaea and Bacteria?Syst Appl Microbiol. 2010; 33:175-182 View ArticlePubMed
  28. Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M and Tindall BJ. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature. 2009; 462:1056-1060 View ArticlePubMed
  29. List of growth media used at DSMZ: Web Site
  30. Sims D, Brettin T, Detter J, Han C, Lapidus A, Copeland A, Glavina Del Rio T, Nolan M, Chen F and Lucas S. Complete genome sequence of Kytococcus sedentarius type strain (541T). Stand Genomic Sci. 2009; 1:12-20 View Article
  31. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW and Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. Bioinformatics. 2010; 11:119 View ArticlePubMed
  32. Pati A, Ivanova N, Mikhailova N, Ovchinikova G, Hooper SD, Lykidis A and Kyrpides NC. GenePRIMP: A gene prediction improvement ipeline for microbial genomes. Nat Methods. 2010; 7:455-457 View ArticlePubMed
  33. Markowitz VM, Ivanova NN, Chen IMA, Chu K and Kyrpides NC. IMG ER: a system for microbial genome annotation expert review and curation. Bioinformatics. 2009; 25:2271-2278 View ArticlePubMed